Genetic or epigenetic inactivation of DNA mismatch repair in tumor precursor cells causes a profound mutator phenotype. The microsatellite mutator phenotype (MMP) was discovered by the detection of deletion/insertion mutations in repeated sequences due to slippage by strand misalignment during DNA replication. The importance of microsatellite sequences for the development of cancer has been well illustrated through the MMP mechanism: cancerrelated genes that contain microsatellite repeats in their coding regions are specifically mutated in MMP-positive tumors. The role of non-coding microsatellites in MMP cancer pathogenesis has only been suggested by scarce experimental data and theoretical speculations. The working hypothesis of this proposal is that the MMP inevitably causes mutations in microsatellites located in non-coding regions of cancer-related genes, and that these mutations may affect their expression through modulation of gene transcription, translation, RNA splicing or mRNA stability. These changes in the levels of negative and positive cell growth regulators in turn, will contribute to awake and/or increase the cell neoplastic potential. The goal of the proposed research is to directly test this hypothesis. The unique feature of this hypothesis is that in the MMP cancer pathway these non-coding microsatellite mutations, despite their presumable modest oncogenic potency, may be equally important for tumorigenesis than other more potent coding mutations, because they occur well before. To test this hypothesis, we will: analyze EGFR CA repeat expansion in MMP-positive cancers; estimate the levels of EGFR expression in subclones of MMP cell lines with different repeat lengths, and their apoptotic response by transfection and small interference RNA assays; correlate EGFR repeat expansion with K-ras oncogene mutations and expression of apoptosis-related genes; examine tumor archeology regarding EGFR expression and repeat expansion; and test the oncogenic potential in vivo of EGFR CA repeat expansion by tumorigenicity assays (Specific aim 1). We will determine the effect of deletions in the 3'UTR of the fl-catenin gene in protein expression and in tumor phenotype by transfection and tumorigenicity assays. We will also test the effect in gene expression of deletions in the 3'UTR of the p53 and other identified candidate genes (Specific aim 2). We will also identify other cancer-related genes with regulatory microsatellite repeats and characterize the non-coding microsatellite expression modulation as outlined before for the EGFR and beta-catenin genes. (Specific aim 3). This proposal will provide insights on a novel pathway of cancer gene expression modulation in tumorigenesis.